Major disruptions in the gastrointestinal microbiota, e.g. caused by antibiotics, can in the long run have a dramatic impact on human health, or instantly open the gate for pathogens such as Clostridium difficile, a lethal pathogen which results in over 15,000 deaths per year. These developments, combined with the fact that anti- biotic-resistance has become an urgent health-threat, require alternative strategies to eradicate pathogens in a subtle manner. The overall objective of this application is to develop a food-grade probiotic that is engineered for programmed release of a therapeutic in the gastrointestinal tract to selectively eradicate C. difficile in a strain-specific manner. By syntheic biology approaches, a phasmid is constructed that replicates in the probiotic, and produces engineered virions that are derived from a broad-spectrum C. difficile phage. The engineered virions encode a CRISPR target sequence specific for C. difficile. While the probiotic passages through the gastrointestinal tract, programmed lysis of the probiotic is activated to release the engineered virions in the gut environment to subsequently inject the CRISPR guide in C. difficile, which combined with the native Cas machinery will induce self-destruction of the pathogen. The rationale of the work proposed is that upon successful completion, a biotherapeutic has been developed to eradicate C. difficile from a complex microbial community without the interference of antibiotics. The following specific aims are pursued: 1) In-vitro assembly of a phasmid that produces engineered virions that kill hyper-virulent Clostridium difficile upon injection of CRISPR-tcdA; 2) Optimize biotherapeutic efficacy; 3) Determine the efficacy of our biotherapeutic vehicle to treat an active and recurrent C. difficile infection in a humanized murine C. difficile infection model. The contribution upon completion of the proposed work is expected to be that a biotherapeutic platform has been developed to kill C. difficile by hijacking the cell's native adaptive immune system, CRISPR-Cas. This contribution will be significant because it is expected to have a broad translational impact in the prevention and treatment of a wide-range of bacterial infections. The research proposed in this application is innovative, in our opinion, be- cause it represents a new departure from the status quo by focusing on in-situ delivery of uniform engineered virions to kill Clostridium difficile by sole CRISPR guide injection.